1. Technical Field
The present invention relates to a method and apparatus for separating a desired component from a solution containing a plurality of biochemical components, and particularly to a method and apparatus using selective adsorption and desorption to accomplish such separation.
2. Description of the Prior Art
Biomolecules can be produced by industrial processes that involve the use of biological systems, principally fermentation, followed by downstream processing which includes the subsequent recovery and purification of desirable products.
Methods for removal or separation of undesirable materials for biological extracts are well known. A current survey of available methods can be found in Volume XXII of "Methods in Enzymology" pp. 273-287 and pp. 476-556 (ed. W. E. Jakoby, Academic Press, New York, N.Y.).
Precipitation is one of the oldest methods for recovering and purifying proteins, and it is still commonly used today. Precipitative methods are based on the physical and chemical properties of the proteins in solution and are effected by changing the solution environment. The four precipitation methods which are most widely used are salting-out, precipitation by organic solvents, isoelectric precipitation, and precipitation by polymers.
The precipitated protein particles can be separated from the aqueous solution by a centrifuge operation or by filtration with a filter press or a rotary drum filter. One of the main factors influencing process economics is the size of the particles to be separated. When the particle size is lower than the 1-2 u range, or about the size of E. coli, the conventional filtration and centrifuge operations are replaced by other techniques.
Among the most rapidly advancing areas for biological separation of small particles are the permeable membrane techniques. There are four types of membrane-based separation techniques: microfiltration, ultrafiltration, hyperfiltration or reverse osmosis, and electrodialysis. They are classified according to the partical size to be removed from a solution.
Another important separation technique for protein mixtures is electrophoresis. Separation by this technique depends on the mobility of protein molecules in gel in an applied electric field. It has been widely used as an analytical technique but is not suitable for large-scale separation. There are three electrophoretic methods--simple electrophoresis, isoelectric focusing and isotachophoresis--which have been primarily aimed at improved analysis of protein mixtures.
The most widely used methods for biological separation are based on adsorption. Column chromatography has been used as an analytical tool to identify small quantities of biomolecules and has been scaled up for purifying commercial quantities of these compounds. A description of chromatographic methods is summarized in the "Encyclopedia of Chemical Technology", Volume 5 pp. 418-420 (Kirk Othmer, 2nd Ed. Wiley-Interscience, New York, N.Y.).
Chromatography is conventionally carried out by flowing a fluid through a packed column. The selected packing is usually in the form of spherical beads as the stationary solid phase in the column. As the fluid flows through the column, the desired biological products and impurities in the fluid phase interact differently with the packing to effect the separation. The interaction can be based on the chemical and physical properties of biological products, such as size, ionic charge, configuration, polarity or solubility, which leads to the differences among the various types of chromatography.
The five main types of chromatography in common use today are gel filtration, ion exchange, adsorption, affinity and reversed phase.
Gel filtration chromatography separates biomolecules on the basis of size. The principle is that small molecules will penetrate the pore structure of the beads to a greater extent than larger molecules will, and therefore will experience a longer retention time in the beads. In effect, the small molecules are hindered as they move through the packed column, allowing large molecules to come out first. This technique is also used for desalting or solvent removal. Sephadex gel, made by Pharmacia Chemicals, and Ultrogel by LKB can fractionate biomolecules in the molecular weight range from 300 to 50,000,000.
Ion exchange chromatography is the most widely used chromatographic technique for purification of biological products. It separates biomolecules based on the ionic charge between the biomolecules and the solid particles. This charge can be controlled by the pH and ionic strength of a solution. As the liquid phase flows through the column, the biomolecules with a net charge opposite to the charge on the ion exchange beads will bind to the beads. The higher the net charge, the stronger the binding interaction will be. Biomolecules that are neutral, or that carry a charge similar to the charge on the ion exchange beads, will flow out of the packed column with the liquid phase. The bound biomolecules are then dislodged from the ion exchange beads as the pH and ionic strength of the liquid phase are varied. A large variety of types of ion exchange beads have been developed with cellulose, agarose, dextran or synthetic polymer as their bases. Carboxy methyl group and phospho group are attached to the base beads to from cation exchangers. Diethylaminoethyl group, triethylaminoethyl group and diethyl-2-hydroxy propyl aminoethyl group are most commonly used as anion exchangers.
Adsorption chromatography separates biomolecules on the basis of chemical-physical adsorption of the biomolecules to a substrate attached to the solid beads. For example, hydroxyapatite crystals immobilized in a cross-linked agarose gel, HA-Ultrogel by LKB, can be used to fractionate nucleic acid, steroids and phospholipids very similar in molecular weight or charge density.
Affinity chromatography is one of the most promising chromatographic techniques because it offers tremendous resolving power. The separation is based on a biospecific interaction. A ligand, designed specifically for the biomolecules of interest, is attached to the affinity support media usually through a spacer arm. The spacer arm provides flexibility and space within the gel matrix, and helps to provide steric configuration during the binding. Biomolecules such as enzyme, antibody, antigen and hormone have been separated or purified by this technique.
Reversed phase chromatography is a powerful technique in the purification of smaller biomolecules including peptides, nucleotides and amino acids. The solid beads, with a rigid, microparticulate structure of large pore volume, are saturated with appropriate hydrocarbons. The biomolecules in the liquid phase flowing through the packed column are dissolved in the supported hydrocarbons according to hydrophobicity. The least soluble biomolecules would come out of the column first and the most soluble ones would elute the column last. Thus, the separation can be achieved as the protein mixtures/fluid flows through the whole column.
All of the conventional separation processes described above are more suited for analytical and preseparation batch processes. They cannot readily meet the demand for high efficiency mass purification. Increasing the size of columns for industrial purification purposes ends up with high equipment and maintenance cost but limited efficiency.